Unique binding pattern for a lineage of human antibodies with broad reactivity against influenza A virus

Most structurally characterized broadly neutralizing antibodies (bnAbs) against influenza A viruses (IAVs) target the conserved conformational epitopes of hemagglutinin (HA). Here, we report a lineage of naturally occurring human antibodies sharing the same germline gene, VH3-48/VK1-12. These antibodies broadly neutralize the major circulating strains of IAV in vitro and in vivo mainly by binding a contiguous epitope of H3N2 HA, but a conformational epitope of H1N1 HA, respectively. Our structural and functional studies of antibody 28-12 revealed that the continuous amino acids in helix A, particularly N49HA2 of H3 HA, are critical to determine the binding feature with 28-12. In contrast, the conformational epitope feature is dependent on the discontinuous segments involving helix A, the fusion peptide, and several HA1 residues within H1N1 HA. We report that this antibody was initially selected by H3 (group 2) viruses and evolved via somatic hypermutation to enhance the reactivity to H3 and acquire cross-neutralization to H1 (group 1) virus. These findings enrich our understanding of different antigenic determinants of heterosubtypic influenza viruses for the recognition of bnAbs and provide a reference for the design of influenza vaccines and more effective antiviral drugs.


REVIEWER COMMENTS, first round
Reviewer #1 (Remarks to the Author): In this study Sun et al. present a lineage of broadly neutralizing antibodies (Bnabs) that neutralize group 1 and 2 strains. Amongst four members of the lineage that shared the same germ-line VH3-48/VK1-12,Bnab 28-12 was the most potent and further characterized. Employing biochemical and structural analyses, the authors discovered that 28-12 has distinct binding modes for H3 (group 2) and H1 (group 1) viruses, binding a continuos epitope within H3 but requiring conformational binding for H1. Analysis of germline-reverted Ab and intermediate mutants suggests that 28-12 was initially selected against an H3 strain and then evolved to have H1 binding ability. Overall, this is an intriguing result, but there are several aspects that deserve further attention.

Specific points:
A comprehensive comparison to other bnAbs that interact with Helix A would be important to include throughout the mansucript. As the authors particularly underline that their mAb has a long CDRH3 (23aa) they should include bnAb 3I14 in this comparison as this mAb appears to have similar features, including a 23aa CDRH3.
The last part of the introduction contains a very long summary of the results, which are again summarized in the discussion. The actual discussion part is however relatively short. A more in depth discussion how 28-12 compares to existing bnAbs would be important.
To ease the reader, the full IAV strains tested should be introduced on first mentioning and noted which strains are human infecting and which not.
If I understand the presented data correctly, 19 antibodies from this donor were successfully sorted and cloned using. Four of these belonged to the same lineage. This seems a very high proportion. Was the sample collected shortly after vaccination? The authors should try to quantify the contribution of this lineage further eg by bulk heavy chain NGS of HA bait sorted cells. Fig1: The methods and legend do not state which HA as was used as bait. Strain/Group needs to be added.
There is very little information on the donor provided: "Blood was collected from a female volunteer previously inoculated with seasonal split influenza vaccine after she had signed the informed consent form." The authors need to specific the precise vaccine (manufacturer, year, which precise HA antigens were included). Time between vaccination and sampling should be stated. Was this the first (ever) vaccination that the person received? History of vaccination and prior IAV infection should be listed. How was the neutralization capacity of the donor plasma?
Line 115-117: The authors state: "The results showed that 28-2, 28-4, 28-6, and 28-12 exhibited cross-neutralizing activity against H1N1 and H3N2, with IC50 values ranging from 0.6382 μg/ml to 7.358 μg/ml (Fig. 1d)." however, according to figure 1d, Abs 28-8, 28-15, 28-16, 28-19, 28-27, and 28-28 also show cross-neutralization. Why where these not followed up further? So in all only 5 of the 19 analyzed Abs did not show cross neutralization activity. This is quite astonishing that so many of the antibodies are neutralizing. The authors should discuss this and also include published bnAbs as reference in their analysis Figure 1d: the full neutralization data and IC50 should be shown.
To underline the claim that 28-12 recognizes a novel epitope the authors should conduct competition mapping and/ or docking analyses with other bnAbs.

In this study Sun et al. present a lineage of broadly neutralizing antibodies (Bnabs)
that neutralize group 1 and 2 strains. Amongst four members of the lineage that shared the same germ-line VH3-48/VK1-12, Bnab 28-12 was the most potent and further characterized. Employing biochemical and structural analyses, the authors discovered that 28-12 has distinct binding modes for H3 (group 2) and H1 (group 1) viruses, binding a continuos epitope within H3 but requiring conformational binding for H1.
Analysis of germline-reverted Ab and intermediate mutants suggests that 28-12 was initially selected against an H3 strain and then evolved to have H1 binding ability.
Overall, this is an intriguing result, but there are several aspects that deserve further attention.
Specific points: 1, A comprehensive comparison to other bnAbs that interact with Helix A would be important to include throughout the mansucript. As the authors particularly underline that their mAb has a long CDRH3 (23aa) they should include bnAb 3I14 in this comparison as this mAb appears to have similar features, including a 23aa CDRH3.
The last part of the introduction contains a very long summary of the results, which are again summarized in the discussion. The actual discussion part is however relatively short. A more in depth discussion how 28-12 compares to existing bnAbs would be important.

Answer:
Thanks for the reviewer's valuable suggestion. We fully agree with the reviewer that a more in-depth discussion on how 28-12 compares to existing bnAbs would be important. Following the reviewer's suggestion, we have added a comprehensive comparison of 28-12 to other representative bnAbs that interact with helix A, including CR9114, CR6261, 39.29, CT149, FI6v3 and 3I14.
Comparison of 28-12 with previously reported cross-group anti-HA stem bnAbs revealed that these mAbs recognize an overlapping region in HA stem. However, the approaching angle for each mAb towards the HA stem region is different. Compared  Fig. 7 and Fig. 4c-e).
The cross-binding on HA1 and HA2 on one protomer probably makes the epitope on H1 HA more conformational. Consistent with the structural analysis, our biochemical assay revealed that both 28-12 and 3I14, the non-HA1/HA2 spanning mAbs (for the HA primary protomer) showed binding activity to the continuous epitope of H3 peptide 36-57 HA2 , but not to H1 (Supplementary Fig. 8). A critical reason for this unique binding feature could also be attributed to residue N49 HA2 in H3 for both 28-12 and 3I14, which is T49 HA2 in H1 (Fig. 5a). Of noting, 28-12 displayed different germline with 3I14 (V H 3-30) and engaged less epitopes in the adjacent protomer ( Supplementary Fig. 7). Thus, 28-12 represents a unique example among the helix A-targeting bnAbs.
In addition, we also compared the contribution of heavy/light chains and CDR loops on antibody recognition. Similar to 3I14, CT149, FI6v3 and 39.29, 28-12 uses both heavy chain and light chain to recognize HA protein with prominent contributions of the heavy chain ( Supplementary Fig. 9). 28-12 has a long HCDR3, which makes much more extensive interaction with HA than other bnAbs. As the reviewer suggested, we also compared the HCDR3 between 28-12 and 3I14 which both have a long HCDR3 (23aa) in length (Supplementary Fig. 10a). 3I14 HCDR3 mainly forms hydrophobic interactions and hydrogen-bonds with helix A and the fusion peptide of H3 or H6 HAs, which is similar to 28-12 HCDR3 in recognizing H3 HA. In addition, 28-12 HCDR3 also contacts with several HA1 residues of H1 HA on the primary protomer. It seems that the HCDR3 of 28-12 is more significant than 3I14 to recognize HA as it contributes more amino acids to interact with H3N2 HA (12) and H1N1 HA (11), respectively, while 3I14 devotes only 7 HCDR3 amino acids to interact with H1 or H6 HAs ( Supplementary Fig. 10b-c).
In conclusion, 28-12, encoded by the rarely reported germline gene VH3-48 is special as it showed different binding pattern between group 1 H1 and group 2 H3 HAs. The helix A-targeting bnAbs contribute differently to bind HA proteins with overlapping but distinct epitopes and multiple approaching angles towards the HA stem region. For some bnAbs such as 3I14, compared with other structural elements, the longer HCDR3 contributes much more interactions with the epitopes on HA.
Among these bnAbs, 28-12 is unique as it devotes much more HCDR3 amino acids to react with HA. The HCDR3 of 28-12 might be developed as a potential template to produce small proteins or peptide-based antivirals. concurrently are extremely rare with frequency ranging from 0.060% to 0.078% (Reply Table 1). We thus speculate that V H 3-48 &V K 1-12 germline genes engage a low frequency in human B cell repertoire and the donor in this study maybe a special case with a high proportion of this lineage antibodies. Strain/Group needs to be added.

Answer:
We are sorry for the inaccurate description. We used HA proteins from a group 2 strain A/HongKong/01/1968 H3N2 as the bait. The manuscript has been modified. We are sorry for the inaccurate description and we have further modified the description. We collected blood from a female volunteer ~4 weeks after vaccination with the seasonal trivalent influenza vaccine produced by Shanghai Institute of  .1).
Reply Fig.1. The neutralizing activity of the donor's plasma against H1N1 and H3N2 viruses in MDCK cells. 50% neutralizing titers (NT50) was analyzed by GraphPad Prism 6.
Representative data are shown with two replicates from two independent experiments. Results are depicted as the mean ± SD.
Why where these not followed up further? So in all only 7 of the 19 analyzed Abs did not show cross neutralization activity. This is quite astonishing that so many of the antibodies are neutralizing. The authors should discuss this and also include published bnAbs as reference in their analysis. Answer: We didn't follow up the other cross-reactive antibodies due to their relatively lower neutralizing activities compared with antibody 28-12. We mainly focus on 28-2, 28-4, 28-6, and 28-12 as they are unique by engaging the same germline genes, V H 3-48/V K 1-12, which is rarely reported and their neutralizing activities are relatively higher than other antibodies isolated in this project, especially for 28-12.
Regarding why "so many of the antibodies are cross-neutralizing" in this study.  We are sorry for the inaccurate description and we have modified the manuscript.
(Main text, lines 120-121) 10, Line 135 "significant" cannot be used in this context as no statistical test was performed. Answer: Thanks for the reviewer's suggestion and we have deleted "significant" in the

Response to Reviewer 2's comments
In this study, Sun, Liu, Lu and colleagues, describe a novel class of neutralizing antibodies. The authors use binding, neutralization and structural determination experiments to show that these Abs are relatively broad in their binding breadth, that they neutralize H1N! and H3N2 viruses and that they bind the HA stem epitope.
Particularly interesting is the finding that a member of this lineage cross-reacts with an H1 and an H3 HA and that its binding mode is slightly different on a different subtype, as it engages a single alpha-helix on an H3, but it requires a conformational epitope consisting of HA1 and HA2 subdomains.
While the structural information provided is very useful as it compares the binding to both an H1 (group 1 influenza) and an H3 (group 2), the study is limited to a single donor and it is unclear how prevalent this class of antibodies is in the general population. The donor's exposure history is not reported so it is impossible to appreciate whether these antibodies were generated under uncommon antigenic exposure conditions. Additionally, the authors call these antibodies broadly neutralizing, but the panel of viruses/HAs is quite limited. Indeed, the authors show that the Abs bind to heterosubtypic HAs, but binding to historical isolates needs to be demonstrated for the Abs to be defined as broadly neutralizing. Answer: Thanks for the reviewer's comments and valuable suggestions. We have  (Figure 6b-c). These findings indicate that the naive B cell that generated 28-12 was specific for H3 HA and that the enhanced binding activity and the cross-reactivity to H1 HA was acquired through somatic mutations.
Iso-ctrl, isotype control. Representative data are shown from two independent experiments.
Results are depicted as the mean ± SD.
3, Were these antibodies tested for auto/polyreactivity? Stem mAbs are typically prone to polyreactivity (see for ex. PMID: 26631631 and PMID: 30837606) and this needs to be tested.

Answer:
Thanks for the reviewer's suggestion. We performed two experiments to study the polyreactivity of V H 3-48/V K 1-12 antibodies with two polyreactive positive control antibodies, an influenza HA stem mAb CR9114 and a prototypical lupus-associated autoantibody 3H9 10 . From the ELISA based assay, we found the V H 3-48/V K 1-12 mAbs and the germline mAb were not polyreactive to polyinosinic-polycytidylic acid (poly I:C), insulin, and lipopolysaccharide (LPS) compared to CR9114 and 3H9.
Similarly, none of the V H 3-48/V K 1-12 mAbs bound at a significant level to HEp-2 cells by immunofluorescence while the control antibody 3H9 showed higher intensity of immunofluorescence. Although CR9114 is polyreactive, it doesn't appear to be significantly reactive to HEp-2 cells (Supplementary Fig. 3  LD50/mouse. This may be a possible reason.
9, Were the HAs made as HA0 or were they activated? Any differential binding especially for H1 HAs since the epitope spans the HA1/HA2? Answer: That's a good question. The HAs used in our project were made as uncleaved HA0. We conducted additional ELISA-based experiments to study "whether there is any differential binding especially for H1 HAs since the epitope spans the HA1/HA2". The HA0 of both H3N2 and H1N1 can be cleaved under TPCK treatment. Cleavage of HA0 does not impair the binding activity of 28-12 to H3N2 HA, but significantly affects the reactivity to H1N1 HA. We also included other HA stem mAbs CR9114, FI6v3, CT149 and 39.29, whose epitope span the HA1/HA2 on the primary protomer in both H1N1 and H3N2 HAs for comparison. 3I14 was another control mAb with epitopes only in the HA2 region (for the primary protomer) of both H3N2 and H6N2 HAs. The results showed that Cleavage of HA0 doesn't affect the binding activity of all the antibodies tested to H3N2, but obviously impairs the reactivity to H1N1 (Ref Figure 2). We hypothesis that H1N1 HA is more sensitive to protease, which may cause conformational change and destroy the complete epitope in H1 HA, thus resulting in loss of binding to antibodies. However, cleavage of H3N2 HA may not change the overall epitope structure, therefore the cleaved H3N2 HA still maintains the reactivity to antibodies. These differences between H1 and H3 HAs may be attributed to the distinct intrinsic properties of HAs, which is not unique for 28-12, but for most of the HA stem mAbs. Although this point is interesting, we didn't include these data in the revised manuscript as it needs more further study to illustrate the different working mechanisms between H1 and H3 HAs. 10, I don't understand why the authors use the word pseudoatomic for their model.
It's an atomic model from a cryo-EM map at near-atomic resolution. Answer: Thanks for the comment from our reviewer. Traditionally, since the cryo-EM maps were usually limited to intermediate or low resolutions, the models built against those cryo-EM maps were termed "pseudoatomic model". In our original manuscript, we used this traditional name. The reviewer is right, since our cryo-EM maps are now at near-atomic resolutions, we then decide to use "atomic model" in the revised manuscript. As the reviewer interested in "how the authors concluded that these antibodies were initially selected by H3", we conducted additional experiments to figure out the original reactivity of the B cells engaging the germline genes of antibody 28-12.
ELISA-based binding activity of a panel of historical H3 and H1 HAs to different versions of 28-12, in which, all or individual somatic mutations in the VH or VL chain were reverted to the germline. The fully germline version (GHGL) of 28-12 reacted with a panel of tested historical H3 HAs, but failed to recognize the tested historical H1 HAs. H3/H1 HAs binding required somatic mutations in both heavy and light chains (Figure 6b-c). These findings indicate that the naive B cell that generated 28-12 was specific for H3 HA and that the cross-reactivity to H1 HA and the enhanced reactivity to both H1 and H3 were acquired through somatic mutations.  In addition, to investigate how frequently the 28-12 epitope is targeted following vaccination, we tested the binding of sera from 50 donors vaccinated with seasonal trivalent inactivated vaccine in 2020 to peptide 36-57 HA2 of H3N2 and H1N1. Of which, 10 sera showed higher binding activity to the peptide 36-57 HA2 of H3N2 than H1N1. The donor LU, the source of 28-12, in this project was included as a positive control. As N49 HA2 is critical for 28-12 binding to H3N2, mutation (N49T HA2 ) of which significantly destructs this reactivity of antibody 28-12 and sera LU. We only observed a notable reduced binding to H3N2 peptide 36-57 HA2 with N49T mutation of two sera (41 and 47). To assess the importance of 28-12 epitope in controlling virus infection with sera 41 and 47, antibody depletion assays were performed against H3N2 peptide 36-57 HA2 . H3 peptide-depleted sera reduced the ability to neutralize H3N2 virus infection, as compared with the unrelated control peptide-depleted sera ( Supplementary Fig. 11). These data indicate the 28-12 epitope-targeting serum antibodies in vaccinated human donors can be found but exist as a relatively low 3, The authors concluded that H3 would initially select the germline-type antibody of VH3-48/DH2-2/JH6 and VK1-12/JK5. If so, the germline-type antibody also binds to  H1N1 isolates (b). GHGL, germline VH paired with germline VL. HGL, matured VH paired with germline VL. GHL, germline VH paired with matured VL. Iso-ctrl, isotype control. Representative data are shown from at least two independent experiments. Results are depicted as the mean ± SD.